http://2008.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=Liblint2008.igem.org - User contributions [en]2024-03-29T14:26:27ZFrom 2008.igem.orgMediaWiki 1.16.5http://2008.igem.org/Jamboree/Schedule/Practice_sessionsJamboree/Schedule/Practice sessions2008-10-30T04:04:12Z<p>Liblint: </p>
<hr />
<div>== Friday November 7 : Practice Talks sign-up sheet ==<br />
<br />
<br />
Use this sign-up sheet to sign up for a slot on Friday night (November 7) to practice your talk. Note that there will NOT be any A/V (audio/visual) support on staff. All classrooms will be unlocked and you should use them and leave them as you found them. <br />
<br />
There are a limited number of time slots available so please only choose one slot. We cannot match the room that you will ultimately give your presentation in with the practice room. This should, however, give you a chance to practice your talk in a new environment.<br />
<br />
Also, there will also be pre-registration available beginning at 6pm. Conference services will be on-site to pass out team registration boxes (see the [[Jamboree/Compete#Team_boxes | Jamboree compete]] page). <br />
<br />
<br />
(Pizza and refreshments will be available on a first-come first-serve basis)<br />
<br />
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<table class="calendar"><h2 class="date"><a name="Friday Practice">Friday, November 7</a></h2><br />
<thead><br />
<tr><br />
<th width="15%">Time</th><br />
<th>room 123</th><br />
<th>room 124</th><br />
<th>room 141</th><br />
<th>room 144</th><br />
<th>room 155</th><br />
<th>room G449</th><br />
<th>room D463</th><br />
<th>room 261*</th><br />
<th>room 262*</th><br />
<th>room 346*</th><br />
<th>room 397*</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr class="even"><br />
<th>6:00p - 6:30p</th><br />
<td>KULeuven</td><br />
<td>UC Berkeley Tools</td><br />
<td>University of Sheffield</td><br />
<td>Melbourne</td><br />
<td>Imperial College</td><br />
<td>F1</td><br />
<td>G1</td><br />
<td>H1</td><br />
<td>I1</td><br />
<td>J1</td><br />
<td>K1</td><br />
</tr><br />
<tr class="odd"><br />
<th>6:30p - 7:00p</th><br />
<td>Heidelberg</td><br />
<td>HKUSTers</td><br />
<td>IIT madras</td><br />
<td>Slovenia</td><br />
<td>E2</td><br />
<td>F2</td><br />
<td>G2</td><br />
<td>H2</td><br />
<td>I2</td><br />
<td>J2</td><br />
<td>K2</td><br />
</tr><br />
<tr class="even"><br />
<th>7:00p - 7:30p</th><br />
<td>Warsaw</td><br />
<td>UVA</td><br />
<td>UChicago</td><br />
<td>Bologna</td><br />
<td>NYMU-Taipei</td><br />
<td>F3</td><br />
<td>UNIPV-Pavia</td><br />
<td>H3</td><br />
<td>I3</td><br />
<td>J3</td><br />
<td>K3</td><br />
</tr><br />
<tr class="even"><br />
<th>7:30p - 8:00p</th><br />
<td>UCSF</td><br />
<td>Peking</td><br />
<td>Delft UT</td><br />
<td>Calgary WetWare</td><br />
<td>iHKU</td><br />
<td>F4</td><br />
<td>Valencia</td><br />
<td>H4</td><br />
<td>I4</td><br />
<td>J4</td><br />
<td>K4</td><br />
</tr><br />
<tr class="odd"><br />
<th>8:00p - 8:30p</th><br />
<td>Caltech</td><br />
<td>Tsinghua</td><br />
<td>CPU-NanJing</td><br />
<td>Paris</td><br />
<td>Washington</td><br />
<td>F5</td><br />
<td>G5</td><br />
<td>H5</td><br />
<td>I5</td><br />
<td>J5</td><br />
<td>K5</td><br />
</tr><br />
<tr class="even"><br />
<th>8:30p - 9:00p</th><br />
<td>Kyoto</td><br />
<td>Alberta_NINT</td><br />
<td>ULeth</td><br />
<td>UAlberta</td><br />
<td>USTC</td><br />
<td>F6</td><br />
<td>G6</td><br />
<td>H6</td><br />
<td>I6</td><br />
<td>J6</td><br />
<td>K6</td><br />
</tr><br />
<tr class="odd"><br />
<th>9:00p - 9:30p</th><br />
<td>Tianjin</td><br />
<td>Waterloo</td><br />
<td>Lethbridge_CCS</td><br />
<td>Rice University</td><br />
<td>Calgary_Ethics</td><br />
<td>H7</td><br />
<td>Chiba</td><br />
<td>H7</td><br />
<td>I7</td><br />
<td>J7</td><br />
<td>K7</td><br />
</tr><br />
<tr class="even"><br />
<th>9:30p - 10:00p</th><br />
<td>Utah State</td><br />
<td>Michigan</td><br />
<td>C8</td><br />
<td>D8</td><br />
<td>E8</td><br />
<td>F8</td><br />
<td>G8</td><br />
<td>H8</td><br />
<td>I8</td><br />
<td>J8</td><br />
<td>K8</td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br />
<br />
Note that rooms marked with an asterisk (*) are smaller conference rooms throughout the Stata Center. Saturday sessions will not be held in these rooms but in order to accommodate all teams who would like to practice their presentations in the 4-hour period on Friday night, we must open these rooms for practice sessions.</div>Liblinthttp://2008.igem.org/Utah_State/29_October_2008Utah State/29 October 20082008-10-30T03:58:53Z<p>Liblint: New page: Sent completed BioBricks to iGEM</p>
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<div>Sent completed BioBricks to iGEM</div>Liblinthttp://2008.igem.org/Team:Utah_State/PartsTeam:Utah State/Parts2008-10-30T03:58:15Z<p>Liblint: /* BIOBRICKS IN PROGRESS */</p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State|<font color="#ffffff">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#99CCFF">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
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==BIOBRICKS==<br />
Nine BioBricks were planned as extracts from the PHB operon. Five of these were from the PHB promoter and four were from the CAB gene cassette. <br />
<br />
[[Image:PartsUSU.jpg|800px|center]]<br />
<br />
==SUBMITTED BIOBRICKS==<br />
<br />
Three PHB promoter BioBricks were successfully ligated into the pSB1A3 plasmid. These BioBricks were constructed to be tested as promoters for green fluorescent protein to determine their autonomy and effectiveness. These BioBricks are labeled in the BioBrick registry as BBa_K089004, BBa_K089005, and BBa_K089006.<br />
<br />
<br />
[[Image:USUBiobrick1.jpg|320px]][[Image:USUBiobrick2.jpg|320px]][[Image:USUBiobrick3.jpg|320px]]<br />
<br />
Miniprepped plasmid DNA from each of the BioBricks were placed into PCR tubes and labeled with their designated BioBrick number. These were placed in a 50-ml free-standing centrifuge tube and sent to iGEM for further testing and placement in the registry. <br />
<br />
[[Image:Submitted_BioBricks_2.JPG|320px]][[Image:Submitted_BioBricks_USU.JPG|320px]][[Image:Trent_Holding_BioBricks.JPG|320px]]<br />
<br />
==BIOBRICKS IN PROGRESS==<br />
<br />
The two remaining PHB promoter regions have been successfully amplified using PCR and are in process of ligations and transformations.<br />
<br />
[[Image:USUBiobrick4.jpg|450px]][[Image:USUBiobrick5.jpg|450px]]<br />
<br />
Two of the CAB cassette genes, phaA and phaB, have been successfully amplified using PCR and are in process of ligation and transformation. The phaC and phaCAB sequences are in earlier stages. <br />
<br />
[[Image:BiobrickPHA.jpg|440px]]<br />
[[Image:BiobrickPHAB.jpg|440px]]<br />
[[Image:BiobrickPHAC.jpg|430px]]<br />
[[Image:BiobrickPHCAB.jpg|430px]]</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:56:52Z<p>Liblint: /* BioBrick Part Assembly */</p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State|<font color="#ffffff">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#99CCFF">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
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<!--- The Mission, Experiments ---><br />
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<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant ''E. coli'' and in ''C. necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Polyhydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
'''1. '''3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
'''2. '''Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
'''3. '''3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The ''E. coli'' strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent ''E. coli'' cells were used for all transformations, excluding one transformation using a toxin-resistant strain.<br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent ''E. coli'' cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.'''Chen GQ, Wu Q. 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
<br />
'''2.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from ''Alcaligenes eutrophus'' H16. Macromolecules. 19:2860-2864<br />
<br />
'''3.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53<br />
<br />
'''4.''' International Genetically Engineered Machines competition. 15 Jun 2008. 26 Jul 2008. <https://igem.org><br />
<br />
'''5.''' Holmes PA. 1985. Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''6.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208<br />
<br />
'''7.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports<br />
<br />
'''8.''' Lee SY, Choi J. 1999. Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151<br />
<br />
'''9.''' Lee SY. 1996. Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
'''10.''' Poirier Y. 1999. Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
<br />
'''11.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page><br />
<br />
'''12.''' Shetty RP, Endy D, and TF Knight Jr. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5<br />
<br />
'''13.'''Schubert P, Kruger N, and Steinbuchel A. 1991. Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. The Journal of Bacteriology. 173(1): 168-175<br />
<br />
'''15.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:56:40Z<p>Liblint: /* Transformations */</p>
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== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant ''E. coli'' and in ''C. necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Polyhydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
'''1. '''3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
'''2. '''Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
'''3. '''3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The ''E. coli'' strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent ''E. coli'' cells were used for all transformations, excluding one transformation using a toxin-resistant strain.<br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.'''Chen GQ, Wu Q. 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
<br />
'''2.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from ''Alcaligenes eutrophus'' H16. Macromolecules. 19:2860-2864<br />
<br />
'''3.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53<br />
<br />
'''4.''' International Genetically Engineered Machines competition. 15 Jun 2008. 26 Jul 2008. <https://igem.org><br />
<br />
'''5.''' Holmes PA. 1985. Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''6.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208<br />
<br />
'''7.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports<br />
<br />
'''8.''' Lee SY, Choi J. 1999. Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151<br />
<br />
'''9.''' Lee SY. 1996. Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
'''10.''' Poirier Y. 1999. Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
<br />
'''11.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page><br />
<br />
'''12.''' Shetty RP, Endy D, and TF Knight Jr. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5<br />
<br />
'''13.'''Schubert P, Kruger N, and Steinbuchel A. 1991. Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. The Journal of Bacteriology. 173(1): 168-175<br />
<br />
'''15.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:56:28Z<p>Liblint: /* Organisms */</p>
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<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant ''E. coli'' and in ''C. necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Polyhydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
'''1. '''3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
'''2. '''Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
'''3. '''3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The ''E. coli'' strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.'''Chen GQ, Wu Q. 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
<br />
'''2.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from ''Alcaligenes eutrophus'' H16. Macromolecules. 19:2860-2864<br />
<br />
'''3.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53<br />
<br />
'''4.''' International Genetically Engineered Machines competition. 15 Jun 2008. 26 Jul 2008. <https://igem.org><br />
<br />
'''5.''' Holmes PA. 1985. Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''6.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208<br />
<br />
'''7.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports<br />
<br />
'''8.''' Lee SY, Choi J. 1999. Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151<br />
<br />
'''9.''' Lee SY. 1996. Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
'''10.''' Poirier Y. 1999. Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
<br />
'''11.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page><br />
<br />
'''12.''' Shetty RP, Endy D, and TF Knight Jr. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5<br />
<br />
'''13.'''Schubert P, Kruger N, and Steinbuchel A. 1991. Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. The Journal of Bacteriology. 173(1): 168-175<br />
<br />
'''15.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:56:10Z<p>Liblint: </p>
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== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant ''E. coli'' and in ''C. necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Polyhydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
'''1. '''3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
'''2. '''Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
'''3. '''3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.'''Chen GQ, Wu Q. 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
<br />
'''2.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from ''Alcaligenes eutrophus'' H16. Macromolecules. 19:2860-2864<br />
<br />
'''3.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53<br />
<br />
'''4.''' International Genetically Engineered Machines competition. 15 Jun 2008. 26 Jul 2008. <https://igem.org><br />
<br />
'''5.''' Holmes PA. 1985. Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''6.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208<br />
<br />
'''7.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports<br />
<br />
'''8.''' Lee SY, Choi J. 1999. Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151<br />
<br />
'''9.''' Lee SY. 1996. Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
'''10.''' Poirier Y. 1999. Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
<br />
'''11.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page><br />
<br />
'''12.''' Shetty RP, Endy D, and TF Knight Jr. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5<br />
<br />
'''13.'''Schubert P, Kruger N, and Steinbuchel A. 1991. Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. The Journal of Bacteriology. 173(1): 168-175<br />
<br />
'''15.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:55:37Z<p>Liblint: /* PHB Metabolic Pathways */</p>
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<!--- The Mission, Experiments ---><br />
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<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant ''E. coli'' and in ''C. necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Polyhydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
'''1. '''3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
'''2. '''Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
'''3. '''3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.'''Chen GQ, Wu Q. 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
<br />
'''2.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from ''Alcaligenes eutrophus'' H16. Macromolecules. 19:2860-2864<br />
<br />
'''3.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53<br />
<br />
'''4.''' International Genetically Engineered Machines competition. 15 Jun 2008. 26 Jul 2008. <https://igem.org><br />
<br />
'''5.''' Holmes PA. 1985. Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''6.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208<br />
<br />
'''7.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports<br />
<br />
'''8.''' Lee SY, Choi J. 1999. Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151<br />
<br />
'''9.''' Lee SY. 1996. Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
'''10.''' Poirier Y. 1999. Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
<br />
'''11.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page><br />
<br />
'''12.''' Shetty RP, Endy D, and TF Knight Jr. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5<br />
<br />
'''13.'''Schubert P, Kruger N, and Steinbuchel A. 1991. Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. The Journal of Bacteriology. 173(1): 168-175<br />
<br />
'''15.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:55:05Z<p>Liblint: /* Introduction */</p>
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<!--- The Mission, Experiments ---><br />
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<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant ''E. coli'' and in ''C. necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Polyhydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.'''Chen GQ, Wu Q. 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
<br />
'''2.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from ''Alcaligenes eutrophus'' H16. Macromolecules. 19:2860-2864<br />
<br />
'''3.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53<br />
<br />
'''4.''' International Genetically Engineered Machines competition. 15 Jun 2008. 26 Jul 2008. <https://igem.org><br />
<br />
'''5.''' Holmes PA. 1985. Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''6.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208<br />
<br />
'''7.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports<br />
<br />
'''8.''' Lee SY, Choi J. 1999. Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151<br />
<br />
'''9.''' Lee SY. 1996. Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
'''10.''' Poirier Y. 1999. Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
<br />
'''11.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page><br />
<br />
'''12.''' Shetty RP, Endy D, and TF Knight Jr. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5<br />
<br />
'''13.'''Schubert P, Kruger N, and Steinbuchel A. 1991. Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. The Journal of Bacteriology. 173(1): 168-175<br />
<br />
'''15.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:54:52Z<p>Liblint: /* Abstract */</p>
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<!--- The Mission, Experiments ---><br />
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== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant ''E. coli'' and in ''C. necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.'''Chen GQ, Wu Q. 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
<br />
'''2.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from ''Alcaligenes eutrophus'' H16. Macromolecules. 19:2860-2864<br />
<br />
'''3.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53<br />
<br />
'''4.''' International Genetically Engineered Machines competition. 15 Jun 2008. 26 Jul 2008. <https://igem.org><br />
<br />
'''5.''' Holmes PA. 1985. Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''6.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208<br />
<br />
'''7.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports<br />
<br />
'''8.''' Lee SY, Choi J. 1999. Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151<br />
<br />
'''9.''' Lee SY. 1996. Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
'''10.''' Poirier Y. 1999. Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
<br />
'''11.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page><br />
<br />
'''12.''' Shetty RP, Endy D, and TF Knight Jr. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5<br />
<br />
'''13.'''Schubert P, Kruger N, and Steinbuchel A. 1991. Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. The Journal of Bacteriology. 173(1): 168-175<br />
<br />
'''15.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449</div>Liblinthttp://2008.igem.org/Team:Utah_State/TeamTeam:Utah State/Team2008-10-30T03:54:18Z<p>Liblint: /* Logan and USU */</p>
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== '''iGEM 2008 at USU''' ==<br />
[[Image:USU_iGEM.jpg|500px|right]]<br />
|'''TEAM USU BACKGROUND'''<br><br />
Utah State University is proud to be involved in the 2008 iGEM competition for its first year. The 2008 USU iGEM team consists of 4 graduate, 5 undergraduate, and 2 high school students under the supervision of faculty with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Though many project topics were seriously discussed, the team chose to study a method of monitoring polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB operon. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction.<br />
<br />
== '''Team Members''' ==<br />
{|border = "10"<br />
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'''FACULTY ADVISORS:'''<br />
*'''Scott Hinton''': Dean of the College of Engineering, USU<br />
*'''Dr. Charles Miller''': Department of Biological and Irrigation Engineering, USU<br />
*'''Dr. Ronald C. Sims''': Department of Biological and Irrigation Engineering, USU<br />
<br />
'''GRADUATE STUDENTS:''' <br />
*'''Junling Huo''': PhD student, Department of Biological and Irrigation Engineering, USU<br />
*'''Steven Merrigan''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Joseph Camire''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Kirsten Sims''': MS student, Department of Biological and Irrigation Engineering, USU <br />
<br />
'''UNDERGRADUATE STUDENTS:'''<br />
*'''Trent Mortensen''': Department of Biological and Irrigation Engineering, USU <br />
*'''Elisabeth Linton''': Department of Biological and Irrigation Engineering, USU <br />
*'''Daniel Nelson''': Department of Biological and Irrigation Engineering, USU <br />
*'''Rachel Porter''': Department of Biological and Irrigation Engineering, USU <br />
<br />
'''HIGH SCHOOL STUDENTS:'''<br />
*'''Garrett Hinton''': Sky View High School<br />
*'''Matthew Sims''': Logan High School<br />
|<br />
<gallery><br />
Image:Scott_Hinton.jpg|Dean Scott Hinton<br />
Image:Charles-Miller.jpg|Dr. Charlie Miller<br />
Image:Ron_sims.jpg|Dr. Ron Sims<br />
Image:JH.jpg|Junlng Huo<br />
Image:JC.jpg|Joseph Camire<br />
Image:Stephen_Merrigan.JPG|Stephen Merrigan<br />
Image:Libby_linton.jpg|Elisabeth Linton<br />
Image:DN.jpg|Daniel Nelson<br />
Image:rachel_249(2).jpg|Rachel Porter<br />
Image:TM.jpg|Trent Mortensen<br />
Image:Sims.jpg|Kirsten and Matthew Sims<br />
Image:GH.jpg|Garrett Hinton<br />
</gallery><br />
|}<br />
<br />
== '''Team Member Contributions''' ==<br />
*'''Junling Huo''' is a PhD student in Biological Engineering who served as an advisor for the iGEM team. He helped to answer student questions, and guide such experimental activities as primer design, electrophoresis, and DNA purification. His personal research is on ''Rhodobacter sphaeroides'', a photosynthetic bacterium.<br />
<br />
*'''Stephen Merrigan''' is currently working on a MS in Biological Engineering at Utah State studying biodiesel production from algae, and has a background in Microbiology. Stephen's primary role was as an advisor for the project. During the first few months of the project Stephen did background research on project topics. He also helped with lab work when necessary, including DNA purification. Stephen also advised on the wiki construction.<br />
<br />
*'''Joseph Camire''' advised on targeting the amplification of the full phaCAB cassette and phaC gene,optimizing the PCR process and testing of primer sets with and without prefix and suffix regions. His work also involved sequence determination and analysis of the phaCAB cassette contained in the source plasmid. He also guided work on the phaC gene, which included targeting at amplification and preparing the gene for site-directed mutagenesis for removal of a critical restriction enzyme site prior to ligation of a new phaCAB cassette for biobrick construction.<br />
<br />
*'''Kirsten Sims''' is a first-year graduate student at Utah State University in Biological and Irrigation Engineering. She received her Bacherlor's degree from Gonzaga University in Biology. Her research focuses on the development of cellulose-derived biofuel. As a member of the USU iGEM team, she was involved in the development and design of the project, as well as participation in the laboratory procedures. She also contributed to the development of the wiki by providing an abstract of the project.<br />
<br />
*'''Trent Mortensen''' is a finishing senior in Biological Engineering. His main research direction is biomedical in nature, using herbal antibiotics as possible alternatives for disease treatment. As part of the USU iGEM team, he played an active role in the planning of the project, group coordination, advising professor consultation, laboratory work, ordering of materials, lab maintenance, Wiki preparation, documentation, and presentation preparations throughout the course of the project.<br />
<br />
*'''Elisabeth Linton''' is a Biological Engineering student. Her research is focused on polyhydroxyalkanoates. For the iGEM team, Libbie helped in project determination and planning, as well as literature review and topic research. She also carried out work in the laboratory like restriction enzyme digestions, gel electrophoresis, DNA isolation, and bacterial transformations. She was also responsible for coordinating efforts and organizing the wiki.<br />
<br />
*'''Daniel Nelson''' is pursuing a degree in Biological Engineering and his current personal research project is “Omega-3 Fatty Acid Production and Extraction in Schizochytrium limacinum SR21.” He has enjoyed participating in group discussions as project ideas and direction have been determined. In the lab, he has worked with the team to isolate, purify, and analyze the key DNA promoters for the PHB synthesis gene. He hopes that through study of these promoter regions, the team will find a way to increase PHB production and create a general gene expression system to monitor cellular product accumulation. Outside of the lab, he has helped to design the logo for the USU team.<br />
<br />
*'''Rachel Porter''' is a sophomore at Utah State University majoring in Biological Engineering/pre-med. For the iGEM project, she helped to carry out some of the lab work. In the lab, she helped isolate and purify DNA, prepare and run electrophoresis gels, and culture cells.<br />
<br />
*'''Matthew Sims''' is a Senior attending Logan High School. This is his first year participating in laboratory research at Utah State University. He plans to go to college next fall and continue his studies in molecular biology and biochemistry. He helped during the summer months to carry out many experiments and obtain and analyze data.<br />
<br />
*'''Garrett Hinton''' is a junior at Sky View High School in Smithfield, Utah. He was heavily involved in the project discussion meetings, as well as the background literature research. Garrett spent many hours working on this project and carried out a substantial portion of the laboratory work.<br />
<br />
'''''We would like to thank our faculty advisers - Dr. Ron Sims, Dean Scott Hinton, and Dr. Charlie Miller - for all of their support and for providing this opportunity for the students.'''''<br />
[[Image:Advisors.jpg|350px|center]]<br />
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== '''Logan and USU''' ==<br />
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<b><a href="http://www.usu.edu/">Utah State University</a></b> is located in Logan, Utah. Logan is about 85 miles north of Salt Lake City, Utah. The city of Logan is located in the heart of Cache Valley near the on the western slopes of the Bear River Mountains, the northernmost branch of the Wasatch Range. The city has a population of approximately 47,000. Logan was established in 1859 and has a rich heritage and wonderful culture. The city of Logan has been stated to be among the safest cities in America.<br><br />
<br><br />
Utah State University was established in 1888 as the Agricultural College of Utah. It's name was later changed to Utah State Agricultural College and was again changed to Utah State University (USU) in 1957. As the land-grant university in Utah, USU conducts world-class research in a variety of agricultural and natural resource disciplines, and has several projects in conjunction with the Department of Defense, NASA. Utah State University also conducts extensive aerospace research. The main campus is located in Logan, Utah. Beyond the Logan campus, Utah State's Extension programs extend academic resources and support throughout the entire state of Utah, having extension locations in each of Utah's 29 counties.<br><br />
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''The Sant Building, shown below, is home to the new USU synthetic biology laboratory''<br><br />
<br />
[[Image:Sant_Blgd.jpg|600px|center]]</div>Liblinthttp://2008.igem.org/Team:Utah_State/TeamTeam:Utah State/Team2008-10-30T03:53:50Z<p>Liblint: /* Team Member Contributions */</p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State|<font color="#ffffff">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#99CCFF">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
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== '''iGEM 2008 at USU''' ==<br />
[[Image:USU_iGEM.jpg|500px|right]]<br />
|'''TEAM USU BACKGROUND'''<br><br />
Utah State University is proud to be involved in the 2008 iGEM competition for its first year. The 2008 USU iGEM team consists of 4 graduate, 5 undergraduate, and 2 high school students under the supervision of faculty with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Though many project topics were seriously discussed, the team chose to study a method of monitoring polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB operon. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction.<br />
<br />
== '''Team Members''' ==<br />
{|border = "10"<br />
|-<br />
|rowspan="3"|<br />
'''FACULTY ADVISORS:'''<br />
*'''Scott Hinton''': Dean of the College of Engineering, USU<br />
*'''Dr. Charles Miller''': Department of Biological and Irrigation Engineering, USU<br />
*'''Dr. Ronald C. Sims''': Department of Biological and Irrigation Engineering, USU<br />
<br />
'''GRADUATE STUDENTS:''' <br />
*'''Junling Huo''': PhD student, Department of Biological and Irrigation Engineering, USU<br />
*'''Steven Merrigan''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Joseph Camire''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Kirsten Sims''': MS student, Department of Biological and Irrigation Engineering, USU <br />
<br />
'''UNDERGRADUATE STUDENTS:'''<br />
*'''Trent Mortensen''': Department of Biological and Irrigation Engineering, USU <br />
*'''Elisabeth Linton''': Department of Biological and Irrigation Engineering, USU <br />
*'''Daniel Nelson''': Department of Biological and Irrigation Engineering, USU <br />
*'''Rachel Porter''': Department of Biological and Irrigation Engineering, USU <br />
<br />
'''HIGH SCHOOL STUDENTS:'''<br />
*'''Garrett Hinton''': Sky View High School<br />
*'''Matthew Sims''': Logan High School<br />
|<br />
<gallery><br />
Image:Scott_Hinton.jpg|Dean Scott Hinton<br />
Image:Charles-Miller.jpg|Dr. Charlie Miller<br />
Image:Ron_sims.jpg|Dr. Ron Sims<br />
Image:JH.jpg|Junlng Huo<br />
Image:JC.jpg|Joseph Camire<br />
Image:Stephen_Merrigan.JPG|Stephen Merrigan<br />
Image:Libby_linton.jpg|Elisabeth Linton<br />
Image:DN.jpg|Daniel Nelson<br />
Image:rachel_249(2).jpg|Rachel Porter<br />
Image:TM.jpg|Trent Mortensen<br />
Image:Sims.jpg|Kirsten and Matthew Sims<br />
Image:GH.jpg|Garrett Hinton<br />
</gallery><br />
|}<br />
<br />
== '''Team Member Contributions''' ==<br />
*'''Junling Huo''' is a PhD student in Biological Engineering who served as an advisor for the iGEM team. He helped to answer student questions, and guide such experimental activities as primer design, electrophoresis, and DNA purification. His personal research is on ''Rhodobacter sphaeroides'', a photosynthetic bacterium.<br />
<br />
*'''Stephen Merrigan''' is currently working on a MS in Biological Engineering at Utah State studying biodiesel production from algae, and has a background in Microbiology. Stephen's primary role was as an advisor for the project. During the first few months of the project Stephen did background research on project topics. He also helped with lab work when necessary, including DNA purification. Stephen also advised on the wiki construction.<br />
<br />
*'''Joseph Camire''' advised on targeting the amplification of the full phaCAB cassette and phaC gene,optimizing the PCR process and testing of primer sets with and without prefix and suffix regions. His work also involved sequence determination and analysis of the phaCAB cassette contained in the source plasmid. He also guided work on the phaC gene, which included targeting at amplification and preparing the gene for site-directed mutagenesis for removal of a critical restriction enzyme site prior to ligation of a new phaCAB cassette for biobrick construction.<br />
<br />
*'''Kirsten Sims''' is a first-year graduate student at Utah State University in Biological and Irrigation Engineering. She received her Bacherlor's degree from Gonzaga University in Biology. Her research focuses on the development of cellulose-derived biofuel. As a member of the USU iGEM team, she was involved in the development and design of the project, as well as participation in the laboratory procedures. She also contributed to the development of the wiki by providing an abstract of the project.<br />
<br />
*'''Trent Mortensen''' is a finishing senior in Biological Engineering. His main research direction is biomedical in nature, using herbal antibiotics as possible alternatives for disease treatment. As part of the USU iGEM team, he played an active role in the planning of the project, group coordination, advising professor consultation, laboratory work, ordering of materials, lab maintenance, Wiki preparation, documentation, and presentation preparations throughout the course of the project.<br />
<br />
*'''Elisabeth Linton''' is a Biological Engineering student. Her research is focused on polyhydroxyalkanoates. For the iGEM team, Libbie helped in project determination and planning, as well as literature review and topic research. She also carried out work in the laboratory like restriction enzyme digestions, gel electrophoresis, DNA isolation, and bacterial transformations. She was also responsible for coordinating efforts and organizing the wiki.<br />
<br />
*'''Daniel Nelson''' is pursuing a degree in Biological Engineering and his current personal research project is “Omega-3 Fatty Acid Production and Extraction in Schizochytrium limacinum SR21.” He has enjoyed participating in group discussions as project ideas and direction have been determined. In the lab, he has worked with the team to isolate, purify, and analyze the key DNA promoters for the PHB synthesis gene. He hopes that through study of these promoter regions, the team will find a way to increase PHB production and create a general gene expression system to monitor cellular product accumulation. Outside of the lab, he has helped to design the logo for the USU team.<br />
<br />
*'''Rachel Porter''' is a sophomore at Utah State University majoring in Biological Engineering/pre-med. For the iGEM project, she helped to carry out some of the lab work. In the lab, she helped isolate and purify DNA, prepare and run electrophoresis gels, and culture cells.<br />
<br />
*'''Matthew Sims''' is a Senior attending Logan High School. This is his first year participating in laboratory research at Utah State University. He plans to go to college next fall and continue his studies in molecular biology and biochemistry. He helped during the summer months to carry out many experiments and obtain and analyze data.<br />
<br />
*'''Garrett Hinton''' is a junior at Sky View High School in Smithfield, Utah. He was heavily involved in the project discussion meetings, as well as the background literature research. Garrett spent many hours working on this project and carried out a substantial portion of the laboratory work.<br />
<br />
'''''We would like to thank our faculty advisers - Dr. Ron Sims, Dean Scott Hinton, and Dr. Charlie Miller - for all of their support and for providing this opportunity for the students.'''''<br />
[[Image:Advisors.jpg|350px|center]]<br />
<br />
== '''Logan and USU''' ==<br />
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</style> <br />
<b><a href="http://www.usu.edu/">Utah State University</a></b> is located in Logan, Utah. Logan is about 85 miles north of Salt Lake City, Utah. The city of Logan is located in the heart of Cache Valley near the on the western slopes of the Bear River Mountains, the northernmost branch of the Wasatch Range. The city has a population of approximately 47,000. Logan was established in 1859 and has a rich heritage and wonderful culture. The city of Logan has been stated to be among the safest cities in America.<br><br />
<br><br />
Utah State University was established in 1888 as the Agricultural College of Utah. It's name was later changed to Utah State Agricultural College and was again changed to Utah State University (USU) in 1957. As the land-grant university in Utah, USU conducts world-class research in a variety of agricultural and natural resource disciplines, and has several projects in conjunction with the Department of Defense, NASA. Utah State University also conducts extensive aerospace research. The main campus is located in Logan, Utah. Beyond the Logan campus, Utah State's Extension programs extend academic resources and support throughout the entire state of Utah, having extension locations in each of Utah's 29 counties.<br><br />
<br />
<br />
</html><br />
''The Sant Building, shown below, is home to the new USU synthetic biology laboratory''<br />
[[Image:Sant_Blgd.jpg|600px|center]]</div>Liblinthttp://2008.igem.org/Team:Utah_State/TeamTeam:Utah State/Team2008-10-30T03:52:46Z<p>Liblint: /* iGEM 2008 at USU */</p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State|<font color="#ffffff">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#99CCFF">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
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body {background-image:url("https://static.igem.org/mediawiki/2008/6/63/Bkgnd14.gif");background-repeat:repeat; }<br />
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{|align="justify"<br />
== '''iGEM 2008 at USU''' ==<br />
[[Image:USU_iGEM.jpg|500px|right]]<br />
|'''TEAM USU BACKGROUND'''<br><br />
Utah State University is proud to be involved in the 2008 iGEM competition for its first year. The 2008 USU iGEM team consists of 4 graduate, 5 undergraduate, and 2 high school students under the supervision of faculty with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Though many project topics were seriously discussed, the team chose to study a method of monitoring polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB operon. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction.<br />
<br />
== '''Team Members''' ==<br />
{|border = "10"<br />
|-<br />
|rowspan="3"|<br />
'''FACULTY ADVISORS:'''<br />
*'''Scott Hinton''': Dean of the College of Engineering, USU<br />
*'''Dr. Charles Miller''': Department of Biological and Irrigation Engineering, USU<br />
*'''Dr. Ronald C. Sims''': Department of Biological and Irrigation Engineering, USU<br />
<br />
'''GRADUATE STUDENTS:''' <br />
*'''Junling Huo''': PhD student, Department of Biological and Irrigation Engineering, USU<br />
*'''Steven Merrigan''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Joseph Camire''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Kirsten Sims''': MS student, Department of Biological and Irrigation Engineering, USU <br />
<br />
'''UNDERGRADUATE STUDENTS:'''<br />
*'''Trent Mortensen''': Department of Biological and Irrigation Engineering, USU <br />
*'''Elisabeth Linton''': Department of Biological and Irrigation Engineering, USU <br />
*'''Daniel Nelson''': Department of Biological and Irrigation Engineering, USU <br />
*'''Rachel Porter''': Department of Biological and Irrigation Engineering, USU <br />
<br />
'''HIGH SCHOOL STUDENTS:'''<br />
*'''Garrett Hinton''': Sky View High School<br />
*'''Matthew Sims''': Logan High School<br />
|<br />
<gallery><br />
Image:Scott_Hinton.jpg|Dean Scott Hinton<br />
Image:Charles-Miller.jpg|Dr. Charlie Miller<br />
Image:Ron_sims.jpg|Dr. Ron Sims<br />
Image:JH.jpg|Junlng Huo<br />
Image:JC.jpg|Joseph Camire<br />
Image:Stephen_Merrigan.JPG|Stephen Merrigan<br />
Image:Libby_linton.jpg|Elisabeth Linton<br />
Image:DN.jpg|Daniel Nelson<br />
Image:rachel_249(2).jpg|Rachel Porter<br />
Image:TM.jpg|Trent Mortensen<br />
Image:Sims.jpg|Kirsten and Matthew Sims<br />
Image:GH.jpg|Garrett Hinton<br />
</gallery><br />
|}<br />
<br />
== '''Team Member Contributions''' ==<br />
*'''Junling Huo''' is a PhD student in Biological Engineering who served as an advisor for the iGEM team. He helped to answer student questions, and guide such experimental activities as primer design, electrophoresis, and DNA purification. His personal research is on ''Rhodobacter sphaeroides'', a photosynthetic bacterium.<br />
<br />
*'''Stephen Merrigan''' is currently working on a MS in Biological Engineering at Utah State studying biodiesel production from algae, and has a background in Microbiology. Stephen's primary role was as an advisor for the project. During the first few months of the project Stephen did background research on project topics. He also helped with lab work when necessary, including DNA purification. Stephen also advised on the wiki construction.<br />
<br />
*'''Joseph Camire''' advised on targeting the amplification of the full phaCAB cassette and phaC gene,optimizing the PCR process and testing of primer sets with and without prefix and suffix regions. His work also involved sequence determination and analysis of the phaCAB cassette contained in the source plasmid. He also guided work on the phaC gene, which included targeting at amplification and preparing the gene for site-directed mutagenesis for removal of a critical restriction enzyme site prior to ligation of a new phaCAB cassette for biobrick construction.<br />
<br />
*'''Kirsten Sims''' is a first-year graduate student at Utah State University in Biological and Irrigation Engineering. She received her Bacherlor's degree from Gonzaga University in Biology. Her research focuses on the development of cellulose-derived biofuel. As a member of the USU iGEM team, she was involved in the development and design of the project, as well as participation in the laboratory procedures. She also contributed to the development of the wiki by providing an abstract of the project.<br />
<br />
*'''Trent Mortensen''' is a finishing senior in Biological Engineering. His main research direction is biomedical in nature, using herbal antibiotics as possible alternatives for disease treatment. As part of the USU iGEM team, he played an active role in the planning of the project, group coordination, advising professor consultation, laboratory work, ordering of materials, lab maintenance, Wiki preparation, documentation, and presentation preparations throughout the course of the project.<br />
<br />
*'''Elisabeth Linton''' is a Biological Engineering student. Her research is focused on the the analysis and verification of gas chromatography and nuclear magnetic resonance methods for the quantification of intracellular polyhydroxyalkanoates in bacterial and environmental samples. For the iGEM team, Libbie helped in project determination and planning, as well as literature review and topic research. She also carried out work in the laboratory like restriction enzyme digestions, gel electrophoresis, DNA isolation, and bacterial transformations. She was also responsible for coordinating efforts and organizing the wiki.<br />
<br />
*'''Daniel Nelson''' is pursuing a degree in Biological Engineering and his current personal research project is “Omega-3 Fatty Acid Production and Extraction in Schizochytrium limacinum SR21.” He has enjoyed participating in group discussions as project ideas and direction have been determined. In the lab, he has worked with the team to isolate, purify, and analyze the key DNA promoters for the PHB synthesis gene. He hopes that through study of these promoter regions, the team will find a way to increase PHB production and create a general gene expression system to monitor cellular product accumulation. Outside of the lab, he has helped to design the logo for the USU team.<br />
<br />
*'''Rachel Porter''' is a sophomore at Utah State University majoring in Biological Engineering/pre-med. For the iGEM project, she helped to carry out some of the lab work. In the lab, she helped isolate and purify DNA, prepare and run electrophoresis gels, and culture cells.<br />
<br />
*'''Matthew Sims''' is a Senior attending Logan High School. This is his first year participating in laboratory research at Utah State University. He plans to go to college next fall and continue his studies in molecular biology and biochemistry. He helped during the summer months to carry out many experiments and obtain and analyze data.<br />
<br />
*'''Garrett Hinton''' is a junior at Sky View High School in Smithfield, Utah. He was heavily involved in the project discussion meetings, as well as the background literature research. Garrett spent many hours working on this project and carried out a substantial portion of the laboratory work.<br />
<br />
'''''We would like to thank our faculty advisers - Dr. Ron Sims, Dean Scott Hinton, and Dr. Charlie Miller - for all of their support and for providing this opportunity for the students.'''''<br />
[[Image:Advisors.jpg|350px|center]]<br />
<br />
== '''Logan and USU''' ==<br />
<html><br />
<style type="text/css"><br />
<br />
</style> <br />
<b><a href="http://www.usu.edu/">Utah State University</a></b> is located in Logan, Utah. Logan is about 85 miles north of Salt Lake City, Utah. The city of Logan is located in the heart of Cache Valley near the on the western slopes of the Bear River Mountains, the northernmost branch of the Wasatch Range. The city has a population of approximately 47,000. Logan was established in 1859 and has a rich heritage and wonderful culture. The city of Logan has been stated to be among the safest cities in America.<br><br />
<br><br />
Utah State University was established in 1888 as the Agricultural College of Utah. It's name was later changed to Utah State Agricultural College and was again changed to Utah State University (USU) in 1957. As the land-grant university in Utah, USU conducts world-class research in a variety of agricultural and natural resource disciplines, and has several projects in conjunction with the Department of Defense, NASA. Utah State University also conducts extensive aerospace research. The main campus is located in Logan, Utah. Beyond the Logan campus, Utah State's Extension programs extend academic resources and support throughout the entire state of Utah, having extension locations in each of Utah's 29 counties.<br><br />
<br />
<br />
</html><br />
''The Sant Building, shown below, is home to the new USU synthetic biology laboratory''<br />
[[Image:Sant_Blgd.jpg|600px|center]]</div>Liblinthttp://2008.igem.org/Team:Utah_StateTeam:Utah State2008-10-30T03:52:17Z<p>Liblint: </p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Utah_State|<font color="#99CCFF">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
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<style type="text/css"><br />
body {background-image:url("https://static.igem.org/mediawiki/2008/6/63/Bkgnd14.gif");background-repeat:repeat; }<br />
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{|align="justify"<br />
[[Image:IGEMBanner.jpg|960 px]]<br />
[[Image:Igemusu.jpg|350px|left|]]<br />
<br />
Utah State University is proud to be involved for the first time in the 2008 iGEM competition. The 2008 USU iGEM team consists of graduate and undergraduate students in Biological Engineering, as well as two high school students, working under the supervision of professors with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Prior to this project, none of the undergraduate or high school students had experience with genetic engineering or synthetic biology. This project provided a great opportunity for these students to learn about synthetic biology and its potential, as well as to gain a great deal of hands on experience with laboratory methods. Through team meetings and working closely together in the lab, team members were also provided with opportunities to get to know each other and the specific project material. <br />
<br />
Though many project topics were discussed, the team chose the project for developing a method of monitoring polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction. This project was carried out from May to October of 2008.<br />
<br />
<br><br />
USU is located in Logan, Utah and is nestled in the beautiful mountains of Cache Valley. For those interested in a great education and are looking for amazing skiing and other outdoor adventures and recreation, USU is perfect!<br />
[[Image:Utah.jpg|470px|left]][[Image:CacheValley.jpg|474px|right]]<br />
<br />
<br />
<br />
|}</div>Liblinthttp://2008.igem.org/Team:Utah_StateTeam:Utah State2008-10-30T03:52:06Z<p>Liblint: </p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Utah_State|<font color="#99CCFF">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
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<style type="text/css"><br />
body {background-image:url("https://static.igem.org/mediawiki/2008/6/63/Bkgnd14.gif");background-repeat:repeat; }<br />
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<br />
{|align="justify"<br />
[[Image:IGEMBanner.jpg|960 px]]<br />
[[Image:Igemusu.jpg|350px|left|]]<br />
<br />
Utah State University is proud to be involved for the first time in the 2008 iGEM competition. The 2008 USU iGEM team consists of graduate and undergraduate students in Biological Engineering, as well as two high school students, working under the supervision of professors with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Prior to this project, none of the undergraduate or high school students had experience with genetic engineering or synthetic biology. This project provided a great opportunity for these students to learn about synthetic biology and its potential, as well as to gain a great deal of hands on experience with laboratory methods. Through team meetings and working closely together in the lab, team members were also provided with opportunities to get to know each other and the specific project material. <br />
<br />
Though many project topics were discussed, the team chose the project for developing a method of monitoring Polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction. This project was carried out from May to October of 2008.<br />
<br />
<br><br />
USU is located in Logan, Utah and is nestled in the beautiful mountains of Cache Valley. For those interested in a great education and are looking for amazing skiing and other outdoor adventures and recreation, USU is perfect!<br />
[[Image:Utah.jpg|470px|left]][[Image:CacheValley.jpg|474px|right]]<br />
<br />
<br />
<br />
|}</div>Liblinthttp://2008.igem.org/Team:Utah_StateTeam:Utah State2008-10-30T03:51:51Z<p>Liblint: </p>
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<div>{| align="center"<br />
|align="center" |[[Image:USUheader2.gif|960 px]]<br><br />
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{|style="font color="#CC3300"; background-color:#212223; cellpadding="3" cellspacing="5" border="2" bordercolor="#cd0000"border-spacing:6px; text-align:center" width="960px"<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Utah_State|<font color="#99CCFF">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
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<br />
Utah State University is proud to be involved for the first time in the 2008 iGEM competition. The 2008 USU iGEM team consists of graduate and undergraduate students in Biological Engineering, as well as two high school students, working under the supervision of professors with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Prior to this project, none of the undergraduate or high school students had experience with genetic engineering or synthetic biology. This project provided a great opportunity for these students to learn about synthetic biology and its potential, as well as to gain a great deal of hands on experience with laboratory methods. Through team meetings and working closely together in the lab, team members were also provided with opportunities to get to know each other and the specific project material. <br />
<br />
Though many project topics were discussed, the team chose the project for developing a method of monitoring Poly-β-hydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction. This project was carried out from May to October of 2008.<br />
<br />
<br><br />
USU is located in Logan, Utah and is nestled in the beautiful mountains of Cache Valley. For those interested in a great education and are looking for amazing skiing and other outdoor adventures and recreation, USU is perfect!<br />
[[Image:Utah.jpg|470px|left]][[Image:CacheValley.jpg|474px|right]]<br />
<br />
<br />
<br />
|}</div>Liblinthttp://2008.igem.org/Team:Utah_StateTeam:Utah State2008-10-30T03:50:36Z<p>Liblint: </p>
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{|style="font color="#CC3300"; background-color:#212223; cellpadding="3" cellspacing="5" border="2" bordercolor="#cd0000"border-spacing:6px; text-align:center" width="960px"<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Utah_State|<font color="#99CCFF">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
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body {background-image:url("https://static.igem.org/mediawiki/2008/6/63/Bkgnd14.gif");background-repeat:repeat; }<br />
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{|align="justify"<br />
[[Image:IGEMBanner.jpg|960 px]]<br />
[[Image:Igemusu.jpg|350px|left|]]<br />
<br />
Utah State University is proud to be involved for the first time in the 2008 iGEM competition. The 2008 USU iGEM team consists of graduate and undergraduate students in Biological Engineering, as well as two high school students, working under the supervision of professors with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Prior to this project, none of the undergraduate or high school students had experience with genetic engineering or synthetic biology. This project provided a great opportunity for these students to learn about synthetic biology and its potential, as well as to gain a great deal of hands on experience with laboratory methods. Through team meetings and working closely together in the lab, team members were also provided with opportunities to get to know each other and the specific project material. <br />
<br />
Though many project topics were seriously discussed, the team chose the project for developing a method of monitoring Polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction. This project was carried out from May to October of 2008.<br />
<br />
<br><br />
USU is located in Logan, Utah and is nestled in the beautiful mountains of Cache Valley. For those interested in a great education and are looking for amazing skiing and other outdoor adventures and recreation, USU is perfect!<br />
[[Image:Utah.jpg|470px|left]][[Image:CacheValley.jpg|474px|right]]<br />
<br />
<br />
<br />
|}</div>Liblinthttp://2008.igem.org/Team:Utah_StateTeam:Utah State2008-10-30T03:49:57Z<p>Liblint: </p>
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<div>{| align="center"<br />
|align="center" |[[Image:USUheader2.gif|960 px]]<br><br />
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{|style="font color="#CC3300"; background-color:#212223; cellpadding="3" cellspacing="5" border="2" bordercolor="#cd0000"border-spacing:6px; text-align:center" width="960px"<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Utah_State|<font color="#99CCFF">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
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body {background-image:url("https://static.igem.org/mediawiki/2008/6/63/Bkgnd14.gif");background-repeat:repeat; }<br />
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</style> <br />
</html><br />
<br />
{|align="justify"<br />
[[Image:IGEMBanner.jpg|960 px]]<br />
[[Image:Igemusu.jpg|350px|left|]]<br />
<br />
Utah State University is proud to be involved for the first time in the 2008 iGEM competition. The 2008 USU iGEM team consists of graduate and undergraduate students in Biological Engineering, as well as two high school students, working under the supervision of professors with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Prior to this project, none of the undergraduate or high school students had experience with genetic engineering or synthetic biology. This project provided a great opportunity for these students to learn about synthetic biology and its potential, as well as to gain a great deal of hands on experience with laboratory methods. Through team meetings and working closely together in the lab, team members were also provided with opportunities to get to know each other and the specific project material. <br />
<br />
Though many project topics were seriously discussed, the team chose the project for developing a method of monitoring Polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction. This project was carried out from May to October of 2008.<br />
<br />
<br><br />
USU is located in Logan, UT and is nestled in the beautiful mountains of Cache Valley. For those interested in a great education and are looking for amazing skiing and other outdoor adventures and recreation, USU is perfect!!<br />
[[Image:Utah.jpg|470px|left]][[Image:CacheValley.jpg|474px|right]]<br />
<br />
<br />
<br />
|}</div>Liblinthttp://2008.igem.org/Team:Utah_StateTeam:Utah State2008-10-30T03:48:58Z<p>Liblint: </p>
<hr />
<div>{| align="center"<br />
|align="center" |[[Image:USUheader2.gif|960 px]]<br><br />
|}<br><br />
{|style="font color="#CC3300"; background-color:#212223; cellpadding="3" cellspacing="5" border="2" bordercolor="#cd0000"border-spacing:6px; text-align:center" width="960px"<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Utah_State|<font color="#99CCFF">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
<html><br />
<style type="text/css"><br />
body {background-image:url("https://static.igem.org/mediawiki/2008/6/63/Bkgnd14.gif");background-repeat:repeat; }<br />
<br />
<br />
</style> <br />
</html><br />
<br />
{|align="justify"<br />
[[Image:IGEMBanner.jpg|960 px]]<br />
[[Image:Igemusu.jpg|350px|left|]]<br />
<br />
Utah State University is proud to be involved for the first time in the 2008 iGEM competition. The 2008 USU iGEM team consists of graduate and undergraduate students in Biological Engineering, as well as two high school students, working under the supervision of professors with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Prior to this project, none of the undergraduate or high school students had experience with genetic engineering or synthetic biology. This project provided a great opportunity for these students to learn about synthetic biology and its potential, as well as to gain a great deal of hands on experience with laboratory methods. Through team meetings and working closely together in the lab, team members were also provided with opportunities to get to know each other and the specific project material. <br />
<br />
Though many project topics were seriously discussed, the team chose the project for developing a method of monitoring Polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction. This project was carried out from May to October of 2008.<br />
<br />
<br><br />
USU is located in Logan, UT and is nestled in the beautiful mountains of Cache Valley. For those interested in a great education and are looking for amazing skiing and other outdoor adventures and recreation, USU is perfect!!<br />
[[Image:Utah.jpg|450px|left]][[Image:CacheValley.jpg|450px|right]]<br />
<br />
<br />
<br />
|}</div>Liblinthttp://2008.igem.org/File:Utah.jpgFile:Utah.jpg2008-10-30T03:48:30Z<p>Liblint: uploaded a new version of "Image:Utah.jpg"</p>
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<div></div>Liblinthttp://2008.igem.org/File:CacheValley.jpgFile:CacheValley.jpg2008-10-30T03:48:18Z<p>Liblint: uploaded a new version of "Image:CacheValley.jpg"</p>
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<div></div>Liblint